Compositions and methods for treating transthyretin (TTR) mediated amyloidosis

ABSTRACT

Disclosed herein are methods for treating hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) in a human patient in need thereof by administering an effective amount of a transthyretin (TTR)-inhibiting composition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Phase of International Application No.PCT/US2018/051796, filed Sep. 19, 2018, which claims the benefit of andpriority to U.S. Provisional Application No. 62/560,667, filed Sep. 19,2017, and U.S. Provisional Application No. 62/561,182, filed Sep. 20,2017, and U.S. Provisional Application No. 62/581,005, filed Nov. 2,2017, which are each hereby incorporated by reference for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically and is hereby incorporated by reference in itsentirety. Said ASCII copy, created Jul. 21, 2023, is named121301-19804_SL.text and is 1,799 bytes in size.

BACKGROUND OF THE INVENTION

Transthyretin (TTR) is a tetrameric protein produced primarily in theliver. Mutations in the TTR gene destabilize the protein tetramer,leading to misfolding of monomers and aggregation into TTR amyloidfibrils (ATTR). Tissue deposition results in systemic ATTR amyloidosis(Coutinho et al., Forty years of experience with type I amyloidneuropathy. Review of 483 cases. In: Glenner et al., Amyloid andAmyloidosis, Amsterdam: Excerpta Media, 1980 pg. 88-93; Hou et al.,Transthyretin and familial amyloidotic polyneuropathy. Recent progressin understanding the molecular mechanism of neurodegeneration. FEBS J2007, 274: 1637-1650; Westermark et al., Fibril in senile systemicamyloidosis is derived from normal transthyretin. Proc Natl Acad Sci USA1990, 87: 2843-2845). Over 100 reported TTR mutations exhibit a spectrumof disease symptoms.

TTR amyloidosis manifests in various forms. When the peripheral nervoussystem is affected more prominently, the disease is termed familialamyloidotic polyneuropathy (FAP). When the heart is primarily involvedbut the nervous system is not, the disease is called familialamyloidotic cardiomyopathy (FAC). A third major type of TTR amyloidosisis called leptomeningeal/CNS (Central Nervous System) amyloidosis.

The most common mutations associated with familial amyloidpolyneuropathy (FAP) and ATTR-associated cardiomyopathy, respectively,are Val30Met (Coelho et al., Tafamidis for transthyretin familialamyloid polyneuropathy: a randomized, controlled trial. Neurology 2012,79: 785-792) and Val122Ile (Connors et al., Cardiac amyloidosis inAfrican Americans: comparison of clinical and laboratory features oftransthyretin V1221 amyloidosis and immunoglobulin light chainamyloidosis. Am Heart J 2009, 158: 607-614).

Current treatment options for FAP focus on stabilizing or decreasing theamount of circulating amyloidogenic protein. Orthotopic livertransplantation reduces mutant TTR levels (Holmgren et al., Biochemicaleffect of liver transplantation in two Swedish patients with familialamyloidotic polyneuropathy (FAP-met30). Clin Genet 1991, 40: 242-246),with improved survival reported in patients with early-stage FAP,although deposition of wild-type TTR may continue (Yazaki et al.,Progressive wild-type transthyretin deposition after livertransplantation preferentially occurs into myocardium in FAP patients.Am J Transplant 2007, 7:235-242; Adams et al., Rapid progression offamilial amyloid polyneuropathy: a multinational natural history studyNeurology 2015 Aug. 25; 85(8) 675-82; Yamashita et al., Long-termsurvival after liver transplantation in patients with familial amyloidpolyneuropathy. Neurology 2012, 78: 637-643; Okamoto et al., Livertransplantation for familial amyloidotic polyneuropathy: impact onSwedish patients' survival. Liver Transpl 2009, 15:1229-1235; Stangou etal., Progressive cardiac amyloidosis following liver transplantation forfamilial amyloid polyneuropathy: implications for amyloidfibrillogenesis. Transplantation 1998, 66:229-233; Fosby et al., Livertransplantation in the Nordic countries—An intention to treat andpost-transplant analysis from The Nordic Liver Transplant Registry1982-2013. Scand J Gastroenterol. 2015 June; 50(6):797-808.Transplantation, in press).

Tafamidis and diflunisal stabilize circulating TTR tetramers, which canslow the rate of disease progression (Berk et al., Repurposingdiflunisal for familial amyloid polyneuropathy: a randomized clinicaltrial. JAMA 2013, 310: 2658-2667; Coelho et al., 2012; Coelho et al.,Long-term effects of tafamidis for the treatment of transthyretinfamilial amyloid polyneuropathy. J Neurol 2013, 260: 2802-2814; Lozeronet al., Effect on disability and safety of Tafamidis in late onset ofMet30 transthyretin familial amyloid polyneuropathy. Eur J Neurol 2013,20: 1539-1545). However, symptoms continue to worsen on treatment in alarge proportion of patients, highlighting the need for new,disease-modifying treatment options for FAP.

Description of dsRNA targeting TTR can be found in, for example,International patent application no. PCT/US2009/061381 (WO2010/048228)and International patent application no. PCT/US2010/055311(WO2011/056883).

SUMMARY

Described herein are methods of treating hereditarytransthyretin-mediated amyloidosis (hATTR amyloidosis) (with or withoutpolyneuropathy and/or cardiomyopathy) in a human patient in needthereof, the method comprising administering to the patient a patisirandrug product as described in Table 1A, 1B, or 1C at a dose of 0.3 mgsiRNA per kg body weight, wherein the patisiran is administeredintravenously once every 3 weeks, wherein the method results instabilization or improvement of a FAP stage, a PND score, a modifiedNeuropathy Impairment Score (mNIS+7) or other neuropathy relatedclinical endpoint, a serum percent TTR concentration, a cardiac markerand/or an echocardiogram parameter.

Also described herein are methods for reducing or arresting an increasein a Neuropathy Impairment Score (NIS) or a modified NIS (mNIS+7) in ahuman subject by administering an effective amount of a transthyretin(TTR)-inhibiting composition, wherein the effective amount reduces aconcentration of TTR protein in serum of the human subject to below 50μg/ml or by at least 80%. Also described herein are methods foradjusting a dosage of a TTR-inhibiting composition for treatment ofincreasing NIS or Familial Amyloidotic Polyneuropathy (FAP) byadministering the TTR-inhibiting composition to a subject having theincreasing NIS or FAP, and determining a level of TTR protein in thesubject having the increasing NIS or FAP. In some embodiments, theamount of the TTR-inhibiting composition subsequently administered tothe subject is increased if the level of TTR protein is greater than 50μg/ml, and the amount of the TTR-inhibiting composition subsequentlyadministered to the subject is decreased if the level of TTR protein isbelow 50 μg/ml. Also described herein are formulated versions of a TTRinhibiting siRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the relationship between progression inΔNIS or ΔmNIS+7 and TTR concentration.

FIG. 2 is a graph illustrating the relationship between progression inΔNIS or ΔmNIS+7 and TTR concentration.

FIG. 3 is the structural formula of the sense and antisense strands ofpatisiran.

FIG. 4 is a graph illustrating an improvement in neurologic impairmentcompared to baseline.

FIG. 5 illustrates the effect of patisiran on mNIS+7.

FIG. 6 illustrates the effect of patisiran on other secondary endpoints.

FIG. 7 is a graph illustrating serum TTR concentration in studyparticipants.

FIG. 8 shows the relationship between serum TTR reduction and mNIS+7score at 18 months.

FIG. 9 shows the shift in both PND score and FAP state at 18 months.

FIG. 10 is a graph showing the results in study participants in the 18month double-blind study treated with patisiran for 12 months.

FIG. 11 is a graph showing the results in study participants in the 24month study.

DETAILED DESCRIPTION

As described in more detail below, disclosed herein are methods oftreating hereditary transthyretin-mediated amyloidosis (hATTRamyloidosis) (with or without polyneuropathy and/or cardiomyopathy) in ahuman patient in need thereof, the method comprising administering tothe patient a patisiran drug product as described in Table 1A, 1B, or 1Cat a dose of 0.3 mg siRNA per kg body weight, wherein the patisiran isadministered intravenously once every 3 weeks, wherein the methodresults in stabilization or improvement of a FAP stage, a PND score, amodified Neuropathy Impairment Score (mNIS+7) or other neuropathyrelated clinical endpoint, a serum percent TTR concentration, a cardiacmarker and/or an echocardiogram parameter. Also disclosed are methodsfor reducing or arresting an increase in a Neuropathy Impairment Score(NIS) or a modified NIS (mNIS+7) in a human subject by administering aneffective amount of a transthyretin (TTR)-inhibiting composition, suchthat the effective amount reduces a concentration of TTR protein inserum to below 50 μg/ml or by at least 80%.

In one embodiment the TTR-inhibiting composition is patisiran, e.g., apatisiran drug product. Patisiran is a small interfering ribonucleicacid (siRNA) which is specific for TTR, formulated in a hepatotropiclipid nanoparticle (LNP) for intravenous (IV) administration.

TTR-Inhibiting Compositions

The methods described herein include administration of TTR-inhibitingcomposition. A TTR-inhibiting composition can be any compound thatreduces a concentration of TTR protein in the serum of a human subject.Examples include but are not limited to RNAi, e.g., siRNA. Examples ofsiRNA include siRNA targeting a TTR gene, e.g., patisirin (described inmore detail) below and revusiran. Examples also include antisense RNA.Examples of antisense RNA targeting a TTR gene can be found in U.S. Pat.No. 8,697,860.

The TTR-inhibiting composition inhibits expression of a TTR gene. Asused herein, “transthyretin” (“TTR”) refers to a gene in a cell. TTR isalso known as ATTR, HsT2651, PALB, prealbumin, TBPA, and transthyretin(prealbumin, amyloidosis type I). The sequence of a human TTR mRNAtranscript can be found at NM_000371. The sequence of mouse TTR mRNA canbe found at NM_013697.2, and the sequence of rat TTR mRNA can be foundat NM_012681.1.

The terms “silence,” “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of” and the like in as far asthey refer to a TTR gene, herein refer to the at least partialsuppression of the expression of a TTR gene, as manifested by areduction of the amount of mRNA which may be isolated from a first cellor group of cells in which a TTR gene is transcribed and which has orhave been treated such that the expression of a TTR gene is inhibited,as compared to a second cell or group of cells substantially identicalto the first cell or group of cells but which has or have not been sotreated (control cells). The degree of inhibition is usually expressedin terms of

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to TTR geneexpression, e.g., the amount of protein encoded by a TTR gene which issecreted by a cell, or the number of cells displaying a certainphenotype, e.g., apoptosis. In principle, TTR gene silencing may bedetermined in any cell expressing the target, either constitutively orby genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given dsRNA inhibitsthe expression of a TTR gene by a certain degree and therefore isencompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

RNAi

In some embodiments, the methods described herein use a TTR-inhibitingcomposition that is an RNAi, e.g., an siRNA, e.g., a dsRNA forinhibiting the expression of a TTR gene. In one embodiment, the siRNA isa dsRNA that targets a TTR gene. The dsRNA includes an antisense strandhaving a region of complementarity which is complementary to at least apart of an mRNA formed in the expression of a TTR gene, and where theregion of complementarity is less than 30 nucleotides in length,generally 19-24 nucleotides in length. The dsRNA of the invention canfurther include one or more single-stranded nucleotide overhangs.TTR-inhibiting siRNAs are described in International patent applicationno. PCT/US2009/061381 (WO2010/048228) and International patentapplication no. PCT/US2010/055311 (WO2011/056883), both incorporated byreference herein in their entireties.

In one embodiment, the TTR-inhibiting composition is patisiran,described in more detail below. In another embodiment, theTTR-inhibiting composition is revusiran, an siRNA specific for TTRconjugated to a Trivalent GalNAc carbohydrate cluster. A completedescription of revusiran can be found in international applicationnumber PCT/US2012/065691 and US Patent Publication No. US20140315835,the contents of which are incorporated by reference in their entirety.

A dsRNA includes two RNA strands that are sufficiently complementary tohybridize to form a duplex structure. One strand of the dsRNA (theantisense strand) includes a region of complementarity that issubstantially complementary, and generally fully complementary, to atarget sequence, derived from the sequence of an mRNA formed during theexpression of a TTR gene, the other strand (the sense strand) includes aregion that is complementary to the antisense strand, such that the twostrands hybridize and form a duplex structure when combined undersuitable conditions. The term “antisense strand” refers to the strand ofa dsRNA which includes a region that is substantially complementary to atarget sequence. As used herein, the term “region of complementarity”refers to the region on the antisense strand that is substantiallycomplementary to a sequence, for example a target sequence, as definedherein. Where the region of complementarity is not fully complementaryto the target sequence, the mismatches are most tolerated in theterminal regions and, if present, are generally in a terminal region orregions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′terminus. The term “sense strand,” as used herein, refers to the strandof a dsRNA that includes a region that is substantially complementary toa region of the antisense strand. Generally, the duplex structure isbetween 15 and 80, or 15 and 60 or 15 and 30 or between 25 and 30, orbetween 18 and 25, or between 19 and 24, or between 19 and 21, or 19,20, or 21 base pairs in length. In one embodiment the duplex is 19 basepairs in length. In another embodiment the duplex is 21 base pairs inlength.

Each strand of a dsRNA is generally between 15 and 80 or 15 and 60 or 15and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25nucleotides in length. In other embodiments, each is strand is 25-30nucleotides in length. Each strand of the duplex can be the same lengthor of different lengths. When two different siRNAs are used incombination, the lengths of each strand of each siRNA can be identicalor can differ.

A dsRNA can include one or more single-stranded overhang(s) of one ormore nucleotides. In one embodiment, at least one end of the dsRNA has asingle-stranded nucleotide overhang of 1 to 4, generally 1 or 2nucleotides. In another embodiment, the antisense strand of the dsRNAhas 1-10 nucleotides overhangs each at the 3′ end and the 5′ end overthe sense strand. In further embodiments, the sense strand of the dsRNAhas 1-10 nucleotides overhangs each at the 3′ end and the 5′ end overthe antisense strand.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3, or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding TTR) including a 5′ UTR, an openreading frame (ORF), or a 3′ UTR. For example, a polynucleotide iscomplementary to at least a part of a TTR mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding TTR.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

Modified dsRNA

In some embodiments, the dsRNA used in the methods described herein ischemically modified to enhance stability. The nucleic acids featured inthe invention may be synthesized and/or modified by methods wellestablished in the art, such as those described in “Current protocols innucleic acid chemistry,” Beaucage, S. L. et al. (Eds.), John Wiley &Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein byreference. Specific examples of dsRNA compounds useful in this inventioninclude dsRNAs containing modified backbones or no naturalinternucleoside linkages. As defined in this specification, dsRNAshaving modified backbones include those that retain a phosphorus atom inthe backbone and those that do not have a phosphorus atom in thebackbone. For the purposes of this specification, and as sometimesreferenced in the art, modified dsRNAs that do not have a phosphorusatom in their internucleoside backbone can also be considered to beoligonucleosides.

Modified dsRNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Modified dsRNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or ore or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other suitable dsRNA mimetics, both the sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,a dsRNA mimetic that has been shown to have excellent hybridizationproperties, is referred to as a peptide nucleic acid (PNA). In PNAcompounds, the sugar backbone of a dsRNA is replaced with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Representative U.S.patents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each ofwhich is herein incorporated by reference. Further teaching of PNAcompounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Other embodiments of the invention are dsRNAs with phosphorothioatebackbones and oligonucleosides with heteroatom backbones, and inparticular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂-[known as a methylene(methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂-[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂-] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Preferred dsRNAs comprise one of the following at the 2′ position: OH;F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred dsRNAs comprise one of the following at the 2′ position:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃,SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an dsRNA, or a group for improving thepharmacodynamic properties of an dsRNA, and other substituents havingsimilar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxygroup. A further preferred modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other preferred modifications include 2′-methoxy (2′-OCH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on the dsRNA,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide.DsRNAs may also have sugar mimetics such as cyclobutyl moieties in placeof the pentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

DsRNAs may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Patisiran

In one embodiment the TTR-inhibiting composition is patisiran. Patisiranis a small interfering ribonucleic acid (siRNA) which is specific forTTR, formulated in a hepatotropic lipid nanoparticle (LNP) forintravenous (IV) administration (Akinc A, Zumbuehl A, et al. Acombinatorial library of lipid-like materials for delivery of RNAitherapeutics. Nat Biotechnol. 2008; 26(5):561-569). This TTR siRNA has atarget region within the 3′ UTR region of the TTR gene to ensure andconfirm homology with WT TTR as well as all reported TTR mutations.Following LNP-mediated delivery to the liver, patisiran targets TTR mRNAfor degradation, resulting in the potent and sustained reduction ofmutant and WT TTR protein via the RNAi mechanism.

The TTR siRNA (also known as ALN-18328) consists of a sense strand andan antisense strand with the following sequences; the lower case lettersindicate 2′-O-methyl versions of the nucleotide:

Patisiran Drug Substance SEQ Sequence ID Strand Oligo name 5′ to 3′ NO:AD-18328 sense A-32345 GuAAccAAGAGu 1 AuuccAudTdT AD-18328 antisenseA-32346 AUGGAAuACUCU 2 UGGUuACdTdT

Typically the patisiran drug substance, i.e., the siRNA is in the formof a pharmaceutically acceptable salt. In some embodiments, thepatisiran drug substance is patisiran sodium. The molecular formula ofpatisiran sodium is C₄₁₂H480N₁₄₈Na₄₀O₂₉₀P₄₀ and the molecular weight is14304 Da. The structural formula of the sense and antisense strands arefound in FIG. 3 .

The manufacturing process consists of synthesizing the two single strandoligonucleotides of the duplex by conventional solid phaseoligonucleotide synthesis. After purification the two oligonucleotidesare annealed into the duplex.

The patisiran drug product is a sterile formulation of the TTR siRNAALN-18328 with lipid excipients (DLin-MC3-DMA, DSPC, cholesterol, andPEGr000-C-DMG) in isotonic phosphate buffered saline.

The formulation of the patisiran drug product is shown in Table 1A, 1B,or 1C below; in some embodiments, the concentration or amount of any onecomponent is +/−0.01, 0.05, 0.1, 0.5, 1.0, 5.0, or 10.0% or theconcentration or amount found in the tables:

TABLE 1A Composition of Patisiran Drug Product Patisiran Component,grade Function Concentration (mg/mL) Active ingredient ALN-18328, cGMP2.0 mg/mL excipient; titratable DLin-MC3-DMA aminolipid for(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- interaction with thetetraen-19-yl-4-(dimethylamino) active ingredient butanoate, cGMP; 12.7mg/mL excipient; stability PEG₂₀₀₀-C-DMG of drug product and((R)-methoxy-PEG₂₀₀₀- desired biodistri-carbamoyl-di-O-myristyl-sn-glyceride), bution cGMP; 1.5 mg/mL Structuralintegrity DSPC (1,2-Distearoyl-sn-Glycero-3- of LNP particlesPhosphocholine), cGMP; 3.1 mg/mL Structural integrity Cholesterol,synthetic, cGMP; of LNP particles 5.9 mg/mL Buffer Phosphate bufferedsaline, cGMP; quantum sufficit

TABLE 1B Composition of Patisiran Drug Product, per 1 ml 2 mg ofpatisiran (2.1 mg of patisiran sodium). 13.0 mg of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 1.6 mg of α-(3′-{[1,2-di(myristyloxy)proponoxyy]carbonylamino}propyl)-ωmethoxy,polyoxyethylene (PEG2000-C-DMG), 3.3 mg of1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 6.2 mg of5-cholesten-3β-ol; 3β-hydroxy-5-cholestene (cholesterol) USP, 2.3 mg ofsodium phosphase dibasic heptahydrate USP, 0.2 mg of potassium phosphatemonobasic anhydrous, 8.8 mg of sodium chloride USP Water for Injection.

TABLE 1C Composition of Patisiran Drug Product, per 1 ml 2 mg ofpatisiran (2.1 mg of patisiran sodium).(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA),α-(3′-{[1,2-di(myristyloxy)proponoxyy]carbonylamino}propyl)-ωmethoxy,polyoxyethylene (PEG2000-C-DMG),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 5-cholesten-3β-ol;3β-hydroxy-5-cholestene (cholesterol) USP, sodium phosphase dibasicheptahydrate USP, potassium phosphate monobasic anhydrous, sodiumchloride USP Water for Injection

In some embodiments, the patisiran drug product is provided in acontainer, e.g., a glass vial, with the following amounts per vial:

TABLE 2 Composition of Patisiran Drug Product including per vialPatisiran Component, grade Function Concentration (mg/mL)/Per vial (mg)Active ingredient ALN-18328, cGMP 2.0 mg/mL/11.0 mg excipient;titratable DLin-MC3-DMA (6Z,9Z,28Z,31Z)- aminolipid forheptatriaconta-6,9,28,31- interaction with thetetraen-19-yl-4-(dimethylamino) active ingredient butanoate, cGMP Mol.wt., 642; 12.7 mg/mL/69.6 mg excipient; stability PEG₂₀₀₀-C-DMG of drugproduct and ((R)-methoxy-PEG₂₀₀₀- desired biodistri-carbamoyl-di-O-myristyl-sn-glyceride), bution cGMP Mol. wt., 2510; 1.5mg/mL/8.2 mg Structural integrity DSPC (1,2-Distearoyl-sn-Glycero-3- ofLNP particles Phosphocholine), cGMP Mol. wt., 790; 3.1 mg/mL/17.3 mgStructural integrity Cholesterol, synthetic, cGMP of LNP particles Mol.wt., 387; 5.9 mg/mL/32.2 mg Buffer Phosphate buffered saline, cGMP;quantum sufficit

Patisiran solution for injection contains 2 mg/mL of TTR siRNA drugsubstance. In some embodiments, the patisiran drug product is packagedin 10 mL glass vials with a fill volume of 5.5 mL. In some embodimentsthe patisiran drug product is packaged with 10 mg in 5 ml as a singleuse vial.

In some embodiments, the container closure system consists of a UnitedStates Pharmacopeia/European Pharmacopoeia (USP/EP) Type I borosilicateglass vial, a Teflon-faced butyl rubber stopper, and an aluminumflip-off cap.

Tetramer Stabilizers

In some embodiments, the methods described herein includeco-administration of a tetramer stabilizer with another TTR-inhibitingcomposition.

Tetramer stabilizers are compounds that bind to the TTR protein and actto stabilize the TTR tetramer. Mutations that destabilize the TTRtetramer result in misfiled and aggregated TTR.

Examples of tetramer stabilizers include tafamidis and diflunisal. Bothtafamidis and diflunisal can slow the rate of disease progression (Berket al., Repurposing diflunisal for familial amyloid polyneuropathy: arandomized clinical trial. JAMA 2013, 310: 2658-2667; Coelho et al.,2012; Coelho et al., Long-term effects of tafamidis for the treatment oftransthyretin familial amyloid polyneuropathy. J Neurol 2013, 260:2802-2814; Lozeron et al., Effect on disability and safety of Tafamidisin late onset of Met30 transthyretin familial amyloid polyneuropathy.Eur J Neurol 2013, 20: 1539-1545).

Subjects and Diagnosis

Disclosed herein are methods of treating hereditarytransthyretin-mediated amyloidosis (hATTR amyloidosis) in a humanpatient in need thereof, wherein the patient may have polyneuropathyand/or cardiomyopathy, the method comprising administering to thepatient a patisiran drug product as described in Table 1A, 1B, or 1C ata dose of 0.3 mg siRNA per kg body weight, wherein the patisiran isadministered intravenously once every 3 weeks, wherein the methodresults in stabilization or improvement of a FAP stage, a PND score, amodified Neuropathy Impairment Score (mNIS+7) or other neuropathyrelated clinical endpoints, a serum percent TTR concentration, a cardiacmarker and/or an echocardiogram parameter.

Also disclosed herein are methods for reducing or arresting an increasein a Neuropathy Impairment Score (NIS) or a modified NIS (mNIS+7) in ahuman subject, wherein the human subject has a TTR related disorder. Insome embodiments, the TTR related disorder is one of the diseases causedby mutations in the transthyretin (TTR) gene. In an embodiment, thedisease is TTR amyloidosis, which manifests in various forms such asfamilial amyloid polyneuropathy (FAP), transthyretin-mediatedamyloidosis (ATTR), and symptomatic polyneuropathy. When the peripheralnervous system is affected more prominently, the disease is termed FAP.When the heart is primarily involved but the nervous system is not, thedisease is called familial amyloidotic cardiomyopathy (FAC). A thirdmajor type of TTR amyloidosis is called leptomeningeal/CNS (CentralNervous System) amyloidosis. ATTR affects the autonomic nervous system.

In some embodiments, the human subject with a TTR related disorder has amutant TTR gene. Over 100 reported TTR mutations exhibit a spectrum ofdisease symptoms. The most common mutations associated with FAP andATTR-associated cardiomyopathy, respectively, are Val30Met andVal122Ile. TTR mutations cause misfolding of the protein and acceleratethe process of TTR amyloid formation, and are the most important riskfactor for the development of clinically significant TTR amyloidosis(also called ATTR (amyloidosis-transthyretin type)). More than 85amyloidogenic TTR variants are known to cause systemic familialamyloidosis.

In some embodiments, a human subject is selected to receive treatmentfor any form of TTR amyloidosis if the human subject is an adult (≥18years) with biopsy-proven ATTR amyloidosis and mild-to-moderateneuropathy. In a further embodiment, the human subject also has one ormore of the following: Karnofsky performance status (KPS)≥60%; body massindex (BMI) 17-33 kg/m²; adequate liver and renal function (aspartatetransaminase (AST) and alanine transaminase (ALT)≤2.5×the upper limit ofnormal (ULN), total bilirubin within normal limits, albumin>3 g/dL, andinternational normalized ratio (INR)≤1.2; serum creatinine≤1.5 ULN); andseronegativity for hepatitis B virus and hepatitis C virus.

In another embodiment, a human subject is excluded from treatment if thehuman subject had a liver transplant; had surgery planned during thetreatment; is HIV-positive; had received an investigational drug otherthan tafamidis or diflunisal within 30 days; had a New York HeartAssociation heart failure classification>2; is pregnant or nursing; hadknown or suspected systemic bacterial, viral, parasitic, or fungalinfections; had unstable angina, uncontrolled clinically significantcardiac arrhythmia; or had a prior severe reaction to a liposomalproduct or known hypersensitivity to oligonucleotides.

Neuropathy Impairment Score (NIS)

The methods disclosed herein reduce or arrest an increase in aNeuropathy Impairment Score (NIS) in a human subject by administering atransthyretin (TTR)-inhibiting composition. NIS refers to a scoringsystem that measures weakness, sensation, and reflexes, especially withrespect to peripheral neuropathy. The NIS score evaluates a standardgroup of muscles for weakness (1 is 25% weak, 2 is 50% weak, 3 is 75%weak, 3.25 is movement against gravity, 3.5 is movement with gravityeliminated, 3.75 is muscle flicker without movement, and 4 isparalyzed), a standard group of muscle stretch reflexes (0 is normal, 1is decreased, 2 is absent), and touch-pressure, vibration, jointposition and motion, and pinprick (all graded on index finger and bigtoe: 0 is normal, 1 is decreased, 2 is absent). Evaluations arecorrected for age, gender, and physical fitness.

In one embodiment, the method for reducing a NIS score results in areduction of NIS by at least 10%. In other embodiments, the method scoreresults in a reduction of NIS by at least 5, 10, 15, 20, 25, 30, 40, orby at least 50%. In other embodiments, the method arrests an increasingNIS score, e.g., the method results in a 0% increase of the NIS score.

Methods for determining an NIS in a human subject are well known to oneof skill in the art and can be found is the following:

Dyck, P J et al., Longitudinal assessment of diabetic polyneuropathyusing a composite score in the Rochester Diabetic Neuropathy Studycohort, Neurology 1997. 49(1): pgs. 229-239).

Dyck P J. Detection, characterization, and staging of polyneuropathy:assessed in diabetics. Muscle Nerve. 1988 January; 11(1):21-32.

Modified Neuropathy Impairment Score (mNIS+7)

In some embodiments, the methods disclosed herein reduce or arrest anincrease in a modified Neuropathy Impairment Score (mNIS+7) in a humansubject by administering a transthyretin (TTR)-inhibiting composition.As well known to one of ordinary skill, mNIS+7 refers to a clinicalexam-based assessment of neurologic impairment (NIS) combined withelectrophysiologic measures of small and large nerve fiber function (NCSand QST), and measurement of autonomic function (postural bloodpressure).

The mNIS+7 score is a modification of the NIS+7 score (which representsNIS plus seven tests). NIS+7 analyzes weakness and muscle stretchreflexes. Five of the seven tests include attributes of nerveconduction. These attributes are the peroneal nerve compound muscleaction potential amplitude, motor nerve conduction velocity and motornerve distal latency (MNDL), tibial MNDL, and sural sensory nerve actionpotential amplitudes. These values are corrected for variables of age,gender, height, and weight. The remaining two of the seven tests includevibratory detection threshold and heart rate decrease with deepbreathing.

The mNIS+7 score modifies NIS+7 to take into account the use of SmartSomatotopic Quantitative Sensation Testing, new autonomic assessments,and the use of compound muscle action potential of amplitudes of theulnar, peroneal, and tibial nerves, and sensory nerve action potentialsof the ulnar and sural nerves (Suanprasert, N. et al., Retrospectivestudy of a TTR FAP cohort to modify NIS+7 for therapeutic trials, J.Neurol. Sci., 2014. 344(1-2): pgs. 121-128).

In an embodiment, the method for reducing an mNIS+7 score results in areduction of mNIS+7 by at least 10%. In other embodiments, the methodscore results in a reduction of an mNIS+7 score by at least 5, 10, 15,20, 25, 30, 40, or by at least 50%. In other embodiments, the methodarrests an increasing mNIS+7, e.g., the method results in a 0% increaseof the mNIS+7.

Quality of Life and Neuropathy Related Clinical Endpoints

In some embodiments, the methods disclosed herein stabilize or improve aquality of life and/or a neuropathy related clinical endpoint. Forexample, the methods described herein can or improve or stabilize aquality of life, a motor strength, a disability, a gait speed, anutritional status, and/or an autonomic symptom in a human patienthaving hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis)with or without polyneuropathy and/or cardiomyopathy, the methodcomprising administering to the patient patisiran drug product asdescribed in Table 1A, 1B, or 1C at a dose of 0.3 mg siRNA per kg bodyweight, wherein the patisiran is administered intravenously for onceevery 3 weeks.

In some embodiments, the methods described herein can improve orstabilize at least one neuropathy related clinical endpoint selectedfrom the group consisting of a Norfolk Quality of LifeQuestionnaire-Diabetic Neuropathy (QOL-DN), a NIS-W; a Rasch-builtOverall Disability Scale (R-ODS); a 10-meter walk test (10-MWT); amodified body mass index (mBMI); and a COMPASS-31 score, a human patienthaving hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis)with or without polyneuropathy and/or cardiomyopathy, the methodcomprising administering to the patient patisiran drug product asdescribed in Table 1A, 1B, or 1C at a dose of 0.3 mg siRNA per kg bodyweight, wherein the patisiran is administered intravenously for onceevery 3 weeks.

FAP Stage and PND Score

In some embodiments, the methods described herein stabilize or improve apolyneuropathy disability (PND) score and familial amyloidoticpolyneuropathy (FAP) stage as described herein. PND Score is determinedas follows: PND I: preserved walking, sensory disturbances; PND II:impaired walking but can walk without stick or crutch; PND IIIA: walkwith 1 stick or crutch; PND IIIB: walk with 2 sticks or crutches; PNDIV: confined to wheelchair or bedridden. FAP stage is as follows: FAP I:unimpaired ambulation; FAP II: assistance with ambulation required; FAPIII: wheelchair bound or bedridden.

Serum TTR Protein Concentration

The methods described herein include administering to the human subjectan effective amount of a transthyretin (TTR)-inhibiting composition,e.g., patisiran, wherein the effective amount reduces a concentration ofTTR protein in serum of the human subject to below 50 μg/ml or by atleast 80%. The serum TTR protein concentration can be determineddirectly using any methods known to one of skill in the art, e.g., anantibody based assay, e.g., an ELISAs. Alternatively, the serum TTRprotein concentration can be determined by measuring the amount of TTRmRNA. In further embodiments, the serum TTR protein concentration isdetermined by measuring the concentration of a surrogate, e.g., VitaminA or retinol binding protein (RBP). In one embodiment, the serum TTRprotein concentration is determined using an ELISA assay as described inthe Examples below.

In some embodiments, the concentration of serum TTR protein is reducedto below 50 μg/ml, or to below 40 μg/ml, 25 μg/ml, or 10 μg/ml. In someembodiments, the concentration of serum TTR protein is reduced by 80%,or by 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or by 95%.

Cardiac Markers and Echocardiographic Parameters

In some embodiments, the methods described herein treat a patient is inneed of treatment for hereditary transthyretin-mediated amyloidosis(hATTR amyloidosis) with cardiomyopathy and the method results in animprovement or a stabilization of a cardiac marker and/or anechocardiogram parameter compared to baseline.

An example of a cardiac marker is a serum NT-proBNP concentration.Examples of echocardiogram parameters are a left ventricle (LV) strainor a LV wall thickness.

AUC

AUC refers to the area under the curve of the concentration of acomposition, e.g. TTR, in the plasma of the bloodstream over time aftera dose of a drug, e.g., a TTR-inhibiting composition, is administered toa patient. It is affected by the rate of absorption into and the rate ofremoval of the composition from the patient's blood plasma. As one ofskill in the art knows, AUC can be determined by calculating theintegral of the plasma composition concentration after the drug isadministered. In another aspect, AUC can be predicted using thefollowing formula:Predicted AUC=(D×F)/CL

where D is the dosage concentration, F is a measure of bioavailability,and CL is the predicted rate of clearance. One of skill in the artappreciates that the values for the predicted AUC have an error in therange of ±3- to 4-fold.

In some embodiments, the data for determining AUC is obtained by takingblood samples from the patient at various time intervals afteradministration of the drug. In one aspect, the mean AUC in the patient'splasma after administration of the TTR-inhibiting composition is in therange of about 9000 to about 18000.

It is understood that the plasma concentration of TTR, may varysignificantly between subjects, due to variability with respect tometabolism and/or possible interactions with other therapeutic agents.In accordance with one aspect of the present invention, the blood plasmaconcentration of TTR may vary from subject to subject. Likewise, valuessuch as maximum plasma concentration (Cmax) or time to reach maximumplasma concentration (Tmax) or area under the curve from time zero totime of last measurable concentration (AUCiast) or total area under theplasma concentration time curve (AUC) may vary from subject to subject.Due to this variability, the amount necessary to constitute “atherapeutically effective amount” of a compound, such as, aTTR-inhibiting composition, may vary from subject to subject.

Pharmaceutical Compositions

The methods described herein include administration of a TTR inhibitingcomposition, e.g., an siRNA targeting a TTR gene, e.g., patisiran. Insome embodiments, the TTR inhibiting composition is a pharmaceuticalcomposition.

As used herein, a “pharmaceutical composition” comprises a TTRinhibiting composition and a pharmaceutically acceptable carrier. Theterm “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical, pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intraparenchymal, intrathecal orintraventricular, administration.

The compositions can be delivered in a manner to target a particulartissue, such as the liver (e.g., the hepatocytes of the liver).Pharmaceutical compositions can be delivered by injection directly intothe brain. The injection can be by stereotactic injection into aparticular region of the brain (e.g., the substantia nigra, cortex,hippocampus, striatum, or globus pallidus), or the dsRNA can bedelivered into multiple regions of the central nervous system (e.g.,into multiple regions of the brain, and/or into the spinal cord). ThedsRNA can also be delivered into diffuse regions of the brain (e.g.,diffuse delivery to the cortex of the brain).

In one embodiment, a dsRNA targeting TTR can be delivered by way of acannula or other delivery device having one end implanted in a tissue,e.g., the brain, e.g., the substantia nigra, cortex, hippocampus,striatum, corpus callosum or globus pallidus of the brain. The cannulacan be connected to a reservoir of the dsRNA composition. The flow ordelivery can be mediated by a pump, e.g., an osmotic pump or minipump,such as an Alzet pump (Durect, Cupertino, Calif.). In one embodiment, apump and reservoir are implanted in an area distant from the tissue,e.g., in the abdomen, and delivery is effected by a conduit leading fromthe pump or reservoir to the site of release. Infusion of the dsRNAcomposition into the brain can be over several hours or for severaldays, e.g., for 1, 2, 3, 5, or 7 days or more. Devices for delivery tothe brain are described, for example, in U.S. Pat. Nos. 6,093,180, and5,814,014.

Dosage and Timing

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the TTR-inhibiting compositions encompassed bythe invention can be made using conventional methodologies or on thebasis of in vivo testing using an appropriate animal model, as describedelsewhere herein.

In general, a suitable dose of a pharmaceutical composition of theTTR-inhibiting composition will be in the range of 0.01 to 200.0milligrams per kilogram body weight of the recipient per day, generallyin the range of 1 to 50 mg per kilogram body weight per day.

For example, the TTR-inhibiting composition can be an siRNA, an can beadministered at, 0.01 mg/kg, 0.05 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg,1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.628 mg/kg, 2mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50mg/kg per single dose. In another embodiment, the dosage is between 0.15mg/kg and 0.3 mg/kg. For example, the TTR-inhibiting composition can beadministered at a dose of 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, or 0.3mg/kg. In an embodiment, the TTR-inhibiting composition is administeredat a dose of 0.3 mg/kg.

The pharmaceutical composition (e.g., patisiran) may be administeredonce daily, or once or twice every 5, 10, 15, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 days. The dosage unit can be compounded fordelivery over several days, e.g., using a conventional sustained releaseformulation which provides sustained release of the TTR-inhibitingcomposition over a several day period. Sustained release formulationsare well known in the art and are particularly useful for delivery ofagents at a particular site, such as could be used with the agents ofthe present invention.

In an embodiment, the TTR-inhibiting composition is patisiran, e.g., thepatisiran drug product, and the dosage is 0.3 mg/kg, and wherein thedose is administered once every 21 days or 3 weeks. In some embodiments,the dose, e.g., the effective amount, is administered about every 3weeks or about every 21 days. In another embodiment, the effectiveamount is 0.3 mg/kg and the effective amount is administered once every21 days or 3 weeks via a 70 minute infusion of 1 mL/min for 15 minutesfollowed by 3 mL/min for 55 minutes. In another embodiment, theeffective amount is 0.3 mg/kg and the effective amount is administeredat two doses every 21-28 days via a 60 minute infusion of 3.3 mL/min, orvia a 70 minute infusion of 1.1 mL/min for 15 minutes followed by 3.3mL/min for 55 minutes.

In some embodiments, the method includes administration of patisiran,e.g., the patisran drug product, at a dosage of 0.3 mg siRNA per kg ofbody weight, administered once every 3 weeks by intravenous infusionover approximately 80 minutes. In some embodiments, the method includeadministration of patisiran at a dosage of 0.3 mg siRNA per kg of bodyweight, administered by intravenous infusion over at 3.3 mL/min over 60minutes, or over 70-minute using a micro-dosing regimen (1.1 mL/min for15 minutes) followed by 3.3 mL/min for the remainder of the dose).

A dosage of a TTR-inhibiting composition can be adjusted for treatmentof increasing NIS or FAP by: administering the TTR-inhibitingcomposition and determining a level of TTR protein in the subject. Ifthe level of TTR protein is greater than 50 μg/ml, the amount ofTTR-inhibiting composition subsequently administered to the subject isincreased, and if the level of TTR protein is below 50 μg/ml, the amountof the TTR-inhibiting composition subsequently administered to thesubject is decreased.

TTR-inhibiting compositions can be administered in combination withother known agents effective in treatment of pathological processesmediated by target gene expression. In an embodiment, patisiran isadministered with a tetramer stabilizer such as tafamidis or diflunisal.In any event, the administering physician can adjust the amount andtiming of patisiran and/or tetramer stabilizer administration on thebasis of results observed using standard measures of efficacy known inthe art or described herein.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B(1992).

The terms “patisiran” and “patisiran drug product” are usedinterchangeably in the Examples, and refer to the formulated siRNA asdescribed in the Tables 1A, 1B, and 1C.

Example 1: Safety and Efficacy of Patisiran for TTR Amyloidosis

In a clinical Phase I trial, patisiran was found to reduce TTR levels inpatients over a period of 28 days. The results of this study werepublished in the New England Journal of Medicine (Coelho et al., N EnglJ Med 2013; 369:819-29.) The publication is incorporated by referencefor all purposes. A summary of study design and results are alsopresented as follows.

The trial was multicenter, randomized, single-blind, placebo-controlled,and dose-ranging to evaluate the safety and efficacy of a single dose ofpatisiran in patients with TTR amyloidosis or in healthy adults. Men andwomen between the ages of 18-45 years were eligible for this trial ifthey were healthy (as determined on the basis of a medical history,physical examination, and 12-lead electrocardiography), had a BMI of18.0-31.5, had adequate liver function and blood counts, and did nothave childbearing potential.

Series of participants (four in each series) were randomly assigned toreceive patisiran at doses of 0.01-0.5 mg/kg or placebo (normal saline)in a 3:1 ratio. The patisiran was administered intravenously during aperiod of 15 minutes and 60 minutes, respectively. In the trial,patients received similar premedication the evening before and the dayof infusion to reduce the risk of infusion-related reactions. Thesemedications included dexamethasone, acetaminophen, diphenhydramine orcetirizine, and ranitidine.

Patisiran pharmacodynamics activity was measured as reflected by serumTTR levels, using a validated enzyme-linked immunosorbent assay (ELISA)for total TTR (Charles River Laboratories, Wilmington Mass.). Baselinelevels of TTR, retinol-binding protein, and vitamin A for each patientwere defined as the mean of four measurements before the administrationof the patisiran. Adverse events were monitored from the start of drugadministration through day 28. Safety monitoring also includedhematologic evaluations, blood chemical analyses, and thyroid-functiontests.

The plasma pharmacokinetics of TTR siRNA contained in patisiran wasevaluated by means of a validated ELISA-based hybridization assay. Fordetection and quantification of siRNA, the ATTO-Probe-HPLC assay (lowerlimit of quantification, 1.0 ng per milliliter) (Tandem Laboratories,Salt Lake City Utah) was used. WinNonlin (Pharsight, Princeton N.J.) wasused to determine the pharmacokinetic estimates.

The knockdown of TTR, vitamin A, and retinol-binding protein wasmeasured as compared with baseline levels. (data not shown).

Results

No significant changes in TTR levels (as compared with placebo) wereobserved at the two lowest doses of patisiran. However, substantial TTRknockdown was observed in all participants receiving doses of 0.15-0.5mg/kg (data not shown). TTR knockdown was rapid, potent, and durableacross all three dose levels, with highly significant changes, ascompared with placebo (P<0.001) through day 28. In light of the robustresponse seen at 0.15 and 0.3 mg/kg and modest incremental improvementin response at 0.5 mg/kg, only one participant received the dose of 0.5mg/kg.

There was little variability among participants in the kinetics ofresponse (data not shown), especially at doses of at least 0.3 mg/kg,with more than 50% lowering by day 3, a nadir level by approximately day10, and continued suppression of more than 50% at day 28, with fullrecovery occurring by day 70. Maximum values for TTR knockdown forparticipants receiving 0.15 mg/kg, 0.3 mg/kg, and 0.5 mg/kg were 85.7%,87.6%, and 93.8%, respectively. The average nadirs at doses of 0.15mg/kg and 0.3 mg/kg were 82.3% (95% confidence interval (CI), 67.7-90.3)and 86.8% (95% CI, 83.8-89.3), respectively; these nadirs showed littlevariability among participants when analyzed as either absolute TTRlevels or percent TTR knockdown and were highly significant, as comparedwith placebo (P<0.001) (data not shown).

The degree of knockdown appeared to determine the duration ofsuppression, with mean reductions at day 28 of 56.6% (95% CI, 11.6-78.7)and 67.1% (95% CI, 45.5-80.1) for participants receiving 0.15 mg/kg and0.3 mg/kg, respectively, and a 76.8% reduction at day 28 for the singlepatient receiving 0.5 mg/kg. The TTR knockdown observed in humans at adose of 0.3 mg/kg was virtually identical to that seen in nonhumanprimates at the same dose level (data not shown). These reductions inTTR by patisiran correlated with changes in levels of retinol-bindingprotein and vitamin A (data not shown).

The use of patisiran did not result in any significant changes inhematologic, liver, or renal measurements or in thyroid function, andthere were no drug-related serious adverse events or any study-drugdiscontinuations because of adverse events (data not shown).

The plasma pharmacokinetic profiles of patisiran showed that the valuesfor the peak plasma concentration and for the area under the curvethrough the last day for the TTR siRNA increased in an approximatelydose-proportional manner over the range of doses that were tested (datanot shown).

Specificity of Patisiran

To further demonstrate the specificity of the effect of patisiran, TTRwas also measured in a group of healthy volunteers in a phase 1 trial ofALN-PCS, which contains an siRNA targeting PCSK9 (a target forcholesterol lowering) that is formulated in the same type of lipidnanoparticle used in patisiran. A single dose of 0.4 mg/kg ALN-PCS(so-called control siRNA) had no effect on TTR (data not shown), whichshowed that the effect of patisiran on TTR was due to specific targetingby the siRNA and not a nonspecific effect of the formulation of lipidnanoparticles.

Additional evidence in support of the specificity and mechanism ofaction of the pharmacodynamics effect of patisiran was obtained usingthe 5′ RACE (rapid amplification of complementary DNA ends) assay onblood samples obtained from participants receiving a dose of 0.3 mg/kgto detect the predicted TTR mRNA cleavage product in circulatingextracellular RNA. To collect the blood samples, serum was collectedafter centrifuging clotted blood sample (pre-dose and at 24 hourspost-dose from subjects) at 1200×g for 20 minutes. Serum was centrifugeda second time at 1200×g for 10 minutes to remove floating cellularmaterial and was then frozen. Thawed serum was mixed with lithiumchloride (final concentration 1M) and incubated at 4 degrees C. for 1hour. Samples were spun at 120,000×g for 2 hours at 4 degrees C. topellet RNA, and total RNA was isolated from pellets by Trizol extraction(Life Technologies, Grand Island, N.Y., USA) and isopropanolprecipitation.

To detect the TTR siRNA-mediated cleavage product, the isolated RNA wasused for ligation-mediated RACE PCR using the GeneRacer kit (LifeTechnologies). RNA was ligated to GeneRacer adapter and reversetranscribed using the TTR-specific reverse primer(5′-aatcaagttaaagtggaatgaaaagtgcctttcacag-3′) (SEQ ID NO:3) followed by2 rounds of PCR using the Gene Racer GR5′ forward primer complementaryto the adaptor and the TTR-specific reverse primer(5′-gcctttcacaggaatgllllattgtctctg-3′)) (SEQ ID NO:4). The nested PCRwas carried out with GR5′ nested primer and the TTR-specific reversenested primer (5′-ctctgcctggacttctaacatagcatatgaggtg-3′)) (SEQ ID NO:5).PCR products were cloned using TOPO-Blunt vector (Life Technologies).The cloned inserts were amplified by colony PCR using M13 forward andreverse primers. The amplicons were sequenced with T7 promoter primer atMacrogen sequencing facility. Sequences from 96 clones were aligned tohuman TTR using CLC WorkBench.

TTR mRNA was detected both in predose samples and in samples obtained 24hours after drug administration. Consistent with the RNAi mechanism, thepredicted mRNA cleavage product was absent in the predose samples andpresent in postdose samples in all three participants (data not shown).

A LC/MS/MS assay for the quantification of wild type and mutant TTR inhuman serum was qualified and conducted by Tandem Labs. The serumsamples were digested using chymotrypsin and then processed by proteinprecipitation extraction prior to analysis by LC/MS/MS. The chymotrypticpeptides TTRW-1 representing wild type TTR and V30M-1 representingmutant V30M were monitored according to their unique specificmass-to-charge ratio transitions. Standard calibration curve dataobtained using stable isotope-labeled peptides (TTRW-1-D8 and V30M-1-D8)were used to calculate endogenous peptide fragments (TTRW-1 and V30M-1)in human serum samples. Peak area ratios for the standards (i.e.TTRW-1-D8 over the internal standard TTRW-L1-D16 and V30M-1-D8 overV30M-L1-D16) were used to create a linear calibration curve using 1/×2weighted least-squares regression analysis. The qualified LC/MS/MSmethod achieved a lower limit of quantitation (LLOQ) of 5 ng/ml withstandard curves ranging from 5 to 2500 ng/ml.

Example 2: Multi-Dose Study for Safety and Efficacy of Patisiran Therapyfor Familial Amyloid Polyneuropathy

In this clinical Phase II trial, multiple doses of patisiran wereadministered to patients with TTR-mediated FAP to evaluate the safety,tolerability, pharmacokinetics, and pharmacodynamics of multipleascending intravenous doses of patisiran in these patients. This datawas presented at the International Symposium on Familial AmyloidoticPolyneuropathy (ISFAP) held in November 2013.

Eligible patients were adults (≥18 years) with biopsy-proven ATTRamyloidosis and mild-to-moderate neuropathy; Karnofsky performancestatus (KPS)≥60%; body mass index (BMI) 17-33 kg/m²; adequate liver andrenal function (aspartate transaminase (AST) and alanine transaminase(ALT)≤2.5×the upper limit of normal (ULN), total bilirubin within normallimits, albumin>3 g/dL, and international normalized ratio (INR)≤1.2;serum creatinine≤1.5 ULN); and seronegativity for hepatitis B virus andhepatitis C virus. Patients were excluded if they had a livertransplant; had surgery planned during the study; were HIV-positive; hadreceived an investigational drug other than tafamidis or diflunisalwithin 30 days; had a New York Heart Association heart failureclassification>2; were pregnant or nursing; had known or suspectedsystemic bacterial, viral, parasitic, or fungal infections; had unstableangina, uncontrolled clinically significant cardiac arrhythmia; or had aprior severe reaction to a liposomal product or known hypersensitivityto oligonucleotides.

This was a multi-center, international, open-label, multiple doseescalation Phase II study of patisiran in patients with FAP. Cohorts of3 patients received two doses of patisiran, with each dose administeredas an intravenous (IV) infusion. Cohorts 1-3 received two doses ofpatisiran 0.01, 0.05 and 0.15 mg/kg every four weeks (Q4W),respectively; cohorts 4 and 5 both received two doses of patisiran 0.3mg/kg Q4W. All patients in cohorts 6-9 received two doses of patisiran0.3 mg/kg administered every three weeks (Q3W). All patients receivedpremedication prior to each patisiran infusion consisting ofdexamethasone, paracetamol (acetaminophen), an H2 blocker (e.g.,ranitidine or famotidine), and an H1 blocker (e.g., cetirizine,hydroxyzine or fexofenadine) to reduce the risk of infusion-relatedreactions. Patisiran was administered IV at 3.3 mL/min over 60 minutes,or over 70-minute using a micro-dosing regimen (1.1 mL/min for 15minutes followed by 3.3 mL/min for the remainder of the dose).

Serum levels of total TTR protein were assessed for all patients usingan enzyme-linked immunosorbent assay (ELISA). Additionally, wild-typeand mutant TTR protein were separately and specifically measured inserum for patients with the Val30Met mutation using a proprietary massspectrometry method (Charles River Laboratories, Quebec, Canada). Serumsamples were collected at screening, and on Days: 0, 1, 2, 7, 10, 14,21, 22, 23 (Q3W only); 28, 29 (Q4W only); 30 (Q4W only); 31 (Q3W only);35, 38 (Q4W only) and 42 49, 56, 112 and 208 of follow-up.

Plasma concentration-time profiles were created for TTR siRNA, based onblood samples collected on Day 0 and at the following time points:pre-dose (within 1 hour of planned dosing start), at end of infusion(EOI), at 5, 10 and 30 minutes and at 1, 2, 4, 6, 24, 48, 168, 336, 504(Day 21, Q3W regimen only) and 672 (Day 28, Q4W regimen only) hourspost-infusion. Additional samples were collected on Days 84 and 180 forthe Q4W regimens, and on Days 35, 91 and 187 for the Q3W regimen. Forcohorts 3-9, blood samples on Day 0 at EOI and 2 hours post-infusionwere also analyzed for both free and encapsulated TTR siRNA. Serum TTRsiRNA was analyzed using a validated ATTO-Probe high-performance liquidchromatography (HPLC) assay (Tandem Laboratories, Salt Lake City, Utah,USA). PK analyses were conducted using non-compartmental and/orcompartmental evaluation of TTR siRNA plasma concentration-time data todetermine PK parameter estimates using the validated software programWinNonlin®. Urine samples were analyzed for levels of excreted TTRsiRNA, and renal clearance (CLR) was measured after dosing.

Serum levels of vitamin A and retinol binding protein (RBP) weremeasured by HPLC and nephelometry, respectively, at the same time pointsspecified for total TTR (Biomins Specialized Medical Pathology, Lyon,France).

Means and variances for TTR knockdown from baseline were calculated forthe PP population, with baseline defined as the average of all pre-dosevalues. Analysis of variance (ANOVA) and analysis of covariance (ANCOVA)were used to analyze the PD data (natural log transformed TTR relativeto baseline), with Tukey's post hoc tests of individual pairwisecomparisons (between dose levels). Nadir TTR levels were defined as theminimum level per patient during the 28-day period (21-day period forQ3W group) after each dose administration (first dose, second doseperiods: Days 1-28, 29-56 and Days 1-21, 22-42 for Q4W and Q3W groups,respectively). Relationships between TTR and RBP or vitamin A, relativeto baseline, and the relationship between wild-type and V30M TTR levels,were explored via linear regression. The dose-proportionality of thepatisiran component in PK parameters was evaluated using a power modelanalysis. AEs were coded using the Medical Dictionary for RegulatoryActivities (MedDRA) coding system, version 15.0, and descriptivestatistics provided for AEs, laboratory data, vital signs data, and ECGinterval data. All statistical analyses were performed using SASsoftware, version 9.3 or higher. Efficacy and pharmacodynamics: mean(SD) baseline serum TTR protein levels were similar across the dosecohorts: 272.9 (98.86), 226.5 (12.67), 276.1 (7.65), 242.6 (38.30) and235.5 (44.45) μg/mL for the 0.01, 0.05, 0.15, 0.3 Q4W and 0.3 mg/kg Q3Wdosage groups, respectively.

In comparison to the 0.01 mg/kg dose cohort, a significant reduction inTTR (p<0.001 by post hoc tests after ANCOVA) was observed after thefirst and second doses of patisiran in the 0.3 mg/kg Q4W and Q3Wcohorts. (data not shown) In patients with the Val30Met mutation, a verysimilar degree of knockdown was observed for wild-type and mutant TTR(data not shown). The level of serum TTR knockdown was highly correlatedwith the reduction in circulating level of RBP (r²=0.89, p<10⁻¹⁵) andvitamin A (r²=0.90, p<10⁻¹⁵) (data not shown).

Although patients taking tafamidis or diflunisal had significantlyincreased baseline levels of serum TTR compared with patients not takingstabilizer therapy (p<0.001 by ANOVA) (data not shown), patisiranadministration resulted in a similar degree of TTR knockdown in thesetwo patient groups (data not shown).

Pharmacokinetics: mean concentrations of the patisiran TTR siRNAcomponent decreased after EOI (data not shown), and there was noaccumulation of siRNA following the second dose on Day 21/28.Measurements of encapsulated versus un-encapsulated concentrations ofTTR siRNA after each dose indicated stability of the circulating LNPformulation. For both the first and second doses, the mean values formaximum plasma concentration (Cmax) and area under the plasmaconcentration-time curve from zero to the last measurable time point(AUC0-last) increased in a dose-proportional manner over the dose rangetested. Cmax and AUC0-last after dose 1 and dose 2 were comparable, withno accumulation. The median terminal half-life of patisiran at Days 0and Days 21/28 was 39-59 hours at doses>0.01 mg/kg, and was relativelyunchanged when comparing dose 1 and dose 2 for each dose cohort.

These Phase II data demonstrate that treatment of patients with FAP withpatisiran led to robust, dose-dependent, and statistically significantknockdown of serum TTR protein levels. Mean sustained reduction in TTRof >80% was achieved with two consecutive doses of patisiran 0.3 mg/kgdosed every 3-4 weeks, with a maximum knockdown of 96% achieved in theQ3W group. These knockdown rates are consistent with the rates observedin the single ascending dose, placebo-controlled Phase 1 study ofpatisiran (Coelho et al. 2013a). Evidence from other systemicamyloidotic diseases indicates that as little as 50% reduction of thedisease-causing protein can result in clinical disease improvement orstabilization (Lachmann et al. 2003; Lachmann et al. 2007). The degreeof TTR knockdown with patisiran was not affected by patients takingtafamidis or diflunisal, suggesting that these TTR stabilizer drugs donot interfere with the pharmacologic activity of patisiran. In patientswith the Val30Met mutation, patisiran suppressed production of bothmutated and wild-type TTR; the latter remains amyloidogenic in patientswith late-onset FAP after liver transplantation (Yazaki et al, 2003;Liepnieks et al, 2010).

Example 3: Reduction of Neurological Impairement as Measured by NIS andmNIS+7 by Administering Patisiran

An Open Label Extension (OLE) study was and is performed with FAPpatients using the protocols described in Example 2. Administration ofpatisiran led to a reduction of both NIS and mNIS+7.

FAP patients previously dosed on Phase 2 trial were eligible to rollover onto Phase 2 OLE study. Up to 2 years of dosing were and areperformed, 0.30 mg/kg every 3 weeks, with clinical endpoints evaluatedevery 6 months. The study objectives included effects on neurologicimpairment (mNIS+7 and NIS), quality of life, mBMI, disability,mobility, grip strength, autonomic symptoms, nerve fiber density in skinbiopsies, cardiac involvement (in cardiac subgroup), and serum TTRlevels.

Patient demographics are shown below.

Characteristic Result Number of patients N = 27 (includes 11 patients incardiac subgroup) Median age 64.0 years (range 29-77) Gender 18 males, 9females TTR genotype Val30Met (V30M) = 20 Tyr116Ser (Y116S) = 1 Ser77Tyr(S77Y) = 2 Phe64Leu (F64L) = 1 Ser77Phe (S77F) = 2 Arg54Thr (R54T) = 1FAP stage/PND Stage 1: 24 I: 14 score Stage 2: 3 II: 10 IIIa: 2 IIIb: 1Concurrent tetramer 13 tafamidis, 7 diflunisal, 7 none stabilizer use atbaseline Current tetramer 12 tafamidis, 6 diflunisal, 9 none stabilizeruse¹ Total doses 511 administered Median doses/ 19 (range 13-24) patientto date Mean treatment 12.9 months (range 8.4-16.7) duration

Baseline characteristics included the following:

Characteristic N Mean (range) mNIS + 7a (max impairment: 304) 27 52.9(2.0-122.5) NIS (max impairment: 244) 27 34.8 (4.0-93.4)

As shown in the table below, administration of patisiran resulted inlowering of serum TTR levels. Patisiran achieved sustained serum TTRlowering of approximately 80%, with further nadir of up to 88% betweendoses.

Mean % Day N Knockdown 1 25 21.4 3 25 46.8 7 25 71.1 17 24 77.8 84 2678.1 168 27 80.5 182 27 87.7 231 25 82.4 234 24 87.0 238 24 88.1 248 2586.0 273  22+ 80.7 357 22 81.3 371 18 87.1 462  3 79.2

As shown in the table below, administration of patisiran resulted in achange in mNIS+7 as measured at 6 and 12 months.

mNIS + 7 Change from Baseline to Month 6 (n = 27) Change from Baselineto Month 12 (n = 20) component Mean (SEM) Median (min, max) Mean (SEM)Median (min, max) Total −1.4 (2.06) −2 (−25.38, 22) −2.5 (2.85) −1.5(−29.75, 24) NIS- 0.2 (1.17) 0 (−9.88, 16) −0.5 (0.86) 0 (−10.38, 6)weakness NIS- −0.7 (0.49) 0 (−8, 3) 0.6 (0.43) 0 (−5.5, 4) reflexesQST^(#) −1.1 (1.49) −1.5 (−15, 16) −2.6 (2.35) −2 (−23, 19) NCS Σ5 0.2(0.13) 0 (−1.5, 1.5) −0.1 (0.25) 0 (−2, 3.5) Postural 0 (0.08) 0 (−1, 1)−0.1 (0.11) 0 (−1.5, 05) BP⁺

As shown in the table below, administration of patisiran resulted inchanges in NIS at 6 and 12 months.

NIS Change from Baseline to Month 6 (n = 27) Change from Baseline toMonth 12 (n = 20) component Mean (SEM) Median (range) Mean (SEM) Median(range) Total −0.7 (1.3) −1.0 (−12.9, 12) 0.4 (1.2) −0.8 (−8.4, 11) NIS-0.2 (1.2) 0 (−9.9, 16) −0.5 (0.9) 0 (−10.4 6) weakness NIS- −0.7 (0.5) 0(−8, 3) 0.6 (0.44) 0 (−5.5, 4) reflexes NIS- −0.3 (0.7) 0 (−9.5, 5) 0.4(0.8) 0.5 (−5, 8) sensation

The relationship between progression in ΔNIS or ΔmNIS+7 and TTRconcentration was explored via linear regression as shown in FIG. 1 andFIG. 2 . TTR and average pre-dose trough [TTR] correlated with a changein mNIS+7 at 6 months.

NIS and mNIS+7 were measured at 0, 6, and 12 months. ΔNIS or ΔmNIS+7from 0 to 6 and 0 to 12 months were used as response variables.Predictor variables included two different measures of TTRconcentration: TTR protein concentration area under the curve (“AUC”),and average percent knockdown relative to baseline at Days 84 and 168(for 0-6 month comparisons) and Days 84, 168, 273, and 357 (for 0-12month comparisons).

For both TTR measures, “baseline” was defined as the average of allpre-dose values. TTR AUC was calculated using raw TTR concentrations(μg/mL) and the method of trapezoids, beginning at baseline value(inserted at Day 0) and extending to Day 182 (for 0-6 month comparisons)or Day 357 (for 0-12 month comparisons). Percent knockdown relative tobaseline was calculated at each scheduled timepoint. Linear regressionwas performed and P values associated with the test of the nullhypothesis that no association exists between predictor and responsevariable were reported.

There was a mean change in mNIS+7 and NIS of −2.5 and 0.4 points,respectively, at 12 months compares favorably to the rapid increase(e.g., 10-18 point increase) in mNIS+7 and NIS estimated at 12 monthsfrom prior FAP studies in a patient population with similar baselineNIS. The favorable impact of patisiran on neuropathy impairment scoreprogression correlated with extent of TTR lowering. This demonstratesthat a reduction in serum TTR burden by patisiran leads to a clinicalbenefit in FAP patients.

Example 4: A Single Randomized, Double-Blind, Placebo-Controlled Phase 3Trial of Patisiran in Patients with hATTR Amyloidosis withPolyneuropathy

Study Design:

The efficacy and safety of patisiran was evaluated in a singlerandomized, double-blind, placebo-controlled Phase 3 trial (APOLLO) inpatients with hATTR amyloidosis with polyneuropathy. The primaryefficacy endpoint was change from baseline in the mNIS+7 compositeneurologic impairment score at 18 months. Secondary endpoints includedthe Norfolk QOL-DN quality of life score as well as measures of motorstrength (NIS-W), disability (R-ODS), gait speed (10-meter walk test),nutritional status (mBMI) and autonomic symptoms (COMPASS-31).Exploratory endpoints included cardiac measures in patients withevidence of cardiac involvement at baseline as well as measures ofdermal amyloid burden and nerve fiber density in skin biopsies.

Summary:

APOLLO met its primary endpoint (mNIS+7) and also showed a highlystatistically significant effect on Norfolk QOL-DN and all othersecondary endpoints demonstrating the clinical benefit of patisiran inhATTR amyloidosis with polyneuropathy. More than 50% of patients treatedwith patisiran had improvement of neurologic impairment at 18 monthscompared to baseline.

Protocol Summary:

Patients were treated with patisiran as described above. Briefly,patients received patisiran (see Table 1) at a dose of 0.3 mg of siRNAper kg body weight, administered intravenously every three weeks (Q3W).Patisiran was administered intravenously at, e.g., 3.3 mL/min over 60minutes, or over 70-minute using a micro-dosing regimen (1.1 mL/min for15 minutes followed by 3.3 mL/min for the remainder of the dose).

In some embodiments, patients received premedication, e.g., the eveningbefore and/or the day of, e.g., one hour before administration ofpatisiran infusion to reduce the risk of infusion-related reactions.These medications included dexamethasone, acetaminophen, diphenhydramineor cetirizine, and ranitidine. In some embodiments, the followingpremedication regimen can be used: IV dexamethasone 10 mg, orequivalent; and oral paracetamol/acetaminophen 500 mg, or equivalent;and IV histamine H1 receptor antagonist (H1 blocker): diphenhydramine 50mg, or equivalent other IV H1 blocker or hydroxyzine 25 mg orfexofenadine 30 or 60 mg PO or cetirizine 10 mg PO; and IV histamine H2receptor antagonist (H2 blocker): ranitidine 50 mg or famotidine 20 mg,or equivalent other H2 blocker dose.

Baseline Characteristics:

APOLLO enrolled 225 patients (148 on patisiran and 77 on placebo).Patients were enrolled at 44 sites in 19 countries from North America,Europe, Asia Pacific and Central/South America from December '13-January'16. The majority of patients were Caucasians (72.4%), 74.2% were males,and most were older adults with a median age of 62 (range 24 to 83years). There was a similar proportion of FAP Stage I versus Stage IIpatients, with mean mNIS+7 scores of 80.9 (range 8-165) and 74.6 (range11-153.5) in the patisiran and placebo groups, respectively. TheVal30Met mutation was present in 42.7% of patients compared to 57.3%with non-Val30Met mutations. Echocardiographic evidence of cardiacamyloid involvement was present in 56%, and 52.9% of all patients had ahistory of prior TTR tetramer stabilizer use. Treatment arms werewell-balanced for age, sex, disease stage, baseline mNIS+7, and priorTTR tetramer stabilizer use. The patisiran arm had more Caucasians(76.4% vs 64.9%), a higher proportion of patients with the non-Val30Metmutation (62.2% vs 48.1%) and with echocardiographic evidence of cardiacinvolvement at baseline (cardiac subpopulation, 60.8% vs 46.8%), as wellas more patients enrolled in North America (25% vs 13%).

TTR Genotype

As described above, the Val30Met mutation was present in 42.7% ofpatients compared to 57.3% with non-Val30Met mutations. The non-Val30Metmutations found in patients are listed below.

TABLE X TTR genotypes HIS-88-ARG GLY-47-ALA TYR-78-PHE ASP-38-VALGLU-89-GLN TYR-114-CYS THR-49-ALA LEU-58-HIS PHE-64-LEU GLY-42-ASPGLU-89-LYS PHE-33-LEU ILE-107-VAL THR-59-LYS SER-77-TYR ILE-84-THRSER-77-PHE PHE-44-SER THR-60-ALA GLY-47-VAL GLU-61-LYS SER-52-PROVAL-71-ALA SER-50-ARG ALA-97-SER ASP-38-ALA VAL-122-ILE LYS-35-ASNALA-45-THR ALA-36-PRO GLU-54-GLN GLY-47-GLU GLU-54-ASP SER-50-ILEGLU-42-GLY THR-49-ILE LIE-107-VAL PRO-24-SER

Disease Stages

The stages of FAP are provided in the Table below:

FAP Stage Descriptions Stage Description 0 No symptoms I Unimpairedambulation; mostly mild sensory, motor, and autonomic neuropathy in thelower limbs II Assistance with ambulation required, mostly moderateimpairment progression to the lower limbs, upper limbs, and trunk. IIIWheelchair-bound or bedridden; severe sensory, motor, and autonomicinvolvement of all limbs.

Disposition:

A total of 185 patients completed study treatment, with a greaterproportion of completers on patisiran (92.6% vs. 62.3% in the patisiranand placebo groups, respectively). A total of 193 patients completed thestudy, with a greater proportion of completers on patisiran (93.2% vs.71.4% in the patisiran and placebo groups, respectively).

6 (7.8%) placebo patients exhibited rapid disease progression (mNIS+7increase of ≥24 points along with FAP stage progression, as determinedby the Clinical Adjudication Committee) at 9 months compared to 1 (0.7%)in the patisiran group.

In the placebo group, the primary reason for study treatmentdiscontinuation was subject withdrawal of consent (15.6%) as well asadverse event (9.1%), progressive disease (5.2%) and death (5.2%), whilethe primary reason for study withdrawal was subject withdrawal ofconsent (14.3%) as well as adverse event (7.8%) and death (5.2%).

In the patisiran group, the primary reason for study treatmentdiscontinuation was death (3.4%) as well as adverse event (2%), and theprimary reason for study withdrawal was death (4.1%) and adverse event(1.4%).

Among the 189 patients who completed APOLLO and were potentiallyeligible to enroll onto the global open-label extension study, 186(98.4%) enrolled onto the ongoing global open-label extension study.

Efficacy Summary

Summaries of the results for both mNIS+7 and the secondary endpoints areshown in the tables below.

Endpoint Domain Range Improvement mNIS + 7 Neuropathy 0-304 pointsNegative change Norfolk QoL QoL −4-136 points Negative change NIS-WMotor Strength 0-192 points Negative change R-ODS Disability 0-48 pointsPositive Change 10-MWT Ambulation meter/second (m/s) Positive Change(gait speed) 1-nBMI Nutritional kg/m² × g/L Positive Change statusCOMPASS-31 Autonomic 0-100 points Negative change Symptoms

Placebo Patisiran Difference Primary (n = 77) ( n= 148) (Patisiran −Endpoint LS Mean CFB LS Mean CFB Placebo) p-value mNIS + 7 27.96 −6.03−33.99 9.26 E−24

Difference Secondary Placebo LS Patisiran LS (Patisiran − Endpoints MeanCFB Mean CFB Placebo) p-value Norfolk-QoL 14.4 −6.7 −21.1 1.10E−10 NIS-W17.93 0.05 −17.87 1.40E−13 R-ODS −8.9 0.0 9.0 4.07E−16 10MWT −0.24 0.080.31 1.88E−12 mBMI −119.4 −3.7 115.7 8.83E−11 COMPASS-31 2.24 −5.29−7.53 0.0008

mNIS+7

The study met its primary efficacy endpoint. In the mITT (modifiedIntent To Treat) population, the patisiran group showed an improvementin neurologic impairment at 18 months compared to baseline (mNIS+7 LSmean (SEM) change of −6.0 (1.7) points) while the placebo group showed aworsening of neurologic impairment (mNIS+7 LS mean (SEM) change of +28.0(2.6) points), representing a highly significant reduction in neuropathyprogression (LS mean difference of −34.0 points, 95% CI: −39.9, −28.1,p=9.26E-24) with patisiran compared to placebo. A similar result wasobserved in the PP population. The effect of patisiran was observed asearly as 9 months (LS mean difference of −16.0 points, 95% CI: −20.7,−11.3), and a consistent effect favoring patisiran was seen across allof the components of mNIS+7.

As shown in FIG. 4 , an improvement in neurologic impairment compared tobaseline (mNIS+7 change of <0 points) at 18 months was seen in 56.1%(95% CI: 48.1%, 64.1%) of patients on patisiran compared to only 3.9%(95% CI: 0.0%, 8.2%) on placebo (Odds ratio of 40.0, p=1.82E-15).

As shown in FIG. 5 , the effect of patisiran on mNIS+7 was observedacross all patient subgroups defined by age, sex, ethnicity, geographicregion, TTR genotype, neuropathy severity, disease stage, and prior TTRtetramer stabilizer use.

Maintenance of the efficacy of patisiran was observed in patients overtreatment regimens of 30 and 36 months, as measured by mNIS+7 score.

Secondary Endpoints

Six secondary endpoints also met statistical significance perhierarchical testing.

As shown in FIG. 6 , the mITT population, the LS mean (SEM) change frombaseline at 18 months for Norfolk QOL-DN was −6.7 (1.8) points forpatisiran, representing an improvement in quality of life, compared to+14.4 (2.7) points for placebo, indicating a worsening of quality oflife. The LS mean difference between the treatment groups was −21.1points (95% CI: −27.2, −15.0, p=1.10E-10), demonstrating a significantimprovement in quality of life with patisiran compared to placebo. Asimilar result was observed in the PP population.

As with mNIS+7, the effect of patisiran on Norfolk QOL-DN was seen asearly as 9 months (LS mean difference of −15.0 points, 95% CI: −19.8,−10.2). The effect of patisiran on Norfolk QOL-DN was observed acrossall patient subgroups defined by age, sex, ethnicity, geographic region,TTR genotype, neuropathy severity, disease stage, and prior TTR tetramerstabilizer use.

Patisiran treatment also resulted in a significant improvement overbaseline compared to placebo at 18 months in multiple additionalsecondary endpoints, including: NIS-W (LS mean difference of −17.9points, 95% CI: −22.3, −13.4, p=1.40E-13); R-ODS (LS mean difference of+9.0 points, 95% CI: 7.0, 10.9, p=4.07E-16); 10-meter walk test (LS meandifference of +0.311 m/sec, 95% CI: 0.23, 0.39, p=1.88E-12); BMI (LSmean difference of +115.7 kg/m2×g/L, 95% CI: 82.4, 149.0, p=8.83E-11);and COMPASS-31 (LS mean difference of −7.5 points, 95% CI: −11.9, −3.2,p=0.0008). The improvement was observed across all patient subgroupsdefined by age, sex, ethnicity, geographic region, TTR genotype,neuropathy severity, disease stage, and prior TTR tetramer stabilizeruse.

As shown in the Table below, all of the secondary endpoints achievedstatistical significance at 18 months. Separation was seen at month 9for all secondary endpoints except for COMPASS-31. Reference ranges areas follows: NIS-W: 0 (better)-192 (worse); R-ODS: 0 (worse)-48 (better);COMPASS 31: 0 (better)-100 (worse).

Treatment Difference Placebo Patisiran (Patisiran − Secondary endpoint,LS Mean (n = 77) (n = 148) Placebo) P-Value NIS-W Baseline score 29.0332.69 Change from baseline 18 mos 17.93 0.05 −17.87 1.40 × 10⁻¹³ R-ODSBaseline score, mean 29.8 29.7 Change from baseline at 18 mos −8.9 0.009.0 4.07 × 10⁻¹⁶ 10-MWT Baseline score, mean 0.79 0.80 (meters/secondChange from baseline at 18 mos −0.24 0.08 0.311 1.88 × 10⁻¹² [m/s]) mBMIBaseline score, mean 990 970 (kg/m² × Change from baseline at D546/18−119.4 −3.7 115.7 8.83 × 10⁻¹¹ g/L) mos COMPASS-31 Baseline score, mean30.31 30.61 Change from baseline at 18 mos 2.24 −5.29 −7.53 0.0008

Serum TTR Reduction

The serum TTR concentration was measured in study participants. Averagepercent reduction in serum TTR was 77.7% (min −38%, max 95%) in patientsreceiving patisiran compared to only 5.8% (min −57%, max 43) reductionin placebo. The effect of patisiran on serum TTR was observed acrosspatient subgroups defined by age, gender, genotype, and prior TTRtetramer stabilizer use. Greater TTR reduction also correlated withimproved changes in both mNIS+7 scores, with an R-value of 0.52 (95% CI:−0.62, −0.41), and Norfolk QoL-DN scores, with an R-value of −0.40 (95%CI: −0.51, −0.27). The data is shown in the graph in FIG. 7 .

A greater TTR reduction correlated with improved change in mNIS+7(R-value 0.52 [95% CI: −0.62, −0.41]). A greater TTR reductioncorrelated with improved change in Norfolk QoL-DN (R-value −0.40 [95%CI: −0.51, −0.27]. The graph in FIG. 8 shows the relationship betweenserum TTR reduction and mNIS+7 score at 18 months.

PND Score and FAP Stage

Patients were evaluated for polyneuropathy disability (PND) score andfamilial amyloidotic polyneuropathy (FAP) stage as described herein. PNDScore is determined as follows: PND I: preserved walking, sensorydisturbances; PND II: impaired walking but can walk without stick orcrutch; PND IIIA: walk with 1 stick or crutch; PND IIIB: walk with 2sticks or crutches; PND IV: confined to wheelchair or bedridden. FAPstage is as follows: FAP I: unimpaired ambulation; FAP II: assistancewith ambulation required; FAP III: wheelchair bound or bedridden.

As shown in FIG. 9 , there was a shift in both PND score and FAP stateat 18 months. Treatment with patisiran resulted in either stabilized orimproved PND scores and FAP stage.

Skin Biopsies: Nerve Fiber Density and Dermal Amyloid Content

Voluntary skin biopsies were performed on study participants. Nervefiber density and dermal amyloid content was determined. Approximately50% of placebo patients with baseline skin biopsy did not have 18-monthfollow-up sample due to dropouts. This, plus substantial variability,limited interpretation of results. However, the results showed anattenuated decrease in intra-epidermal nerve fiber density (IENFD) withpatisiran treatment compared to placebo (nominal p-value significant)and no significant change in sweat gland nerve fiber density (SGNFD) ordermal amyloid content with patisiran treatment compared to placebo.(Data not shown).

Maintenance of Patisiran Efficacy

Study participants in the 18 month double-blind study were treated withpatisiran for 12 months. The results are shown in the graph in FIG. 10 ,and demonstrate a maintenance of the patisiran effect on the mNIS+7 over30 months and evidence of efficacy in patients who previously receivedplacebo.

Study participants in the 24 month study were treated with patisiran for12 months. The results are shown in the graph in FIG. 11 , anddemonstrate a maintenance of the patisiran effect on the mNIS+7 over 36months.

Cardiac Subgroup Analysis

The cardiac subpopulation, e.g., patients with cardiomyopathy, isdescribed above. In general, these were patients with a 13 mm or greaterheart all thickness, and no evidence of high blood pressure or heartvalve disease. The cardiac subpopulation consisted of 36 patients(46.8%) in the placebo subpopulation and 90 patients (60.8%) in thepatisiran population. The total number of patients in the cardiacsubpopulations was 126, or 56% of the patients in the study.

Exploratory cardiac related endpoints, e.g., a cardiac marker and/or anechocardiogram parameter, were evaluated in the entire population. Theresults are shown in the Table below.

Cardiac Exploratory Endpoints Placebo Patisiran Treatment DifferenceNominal Exploratory endpoint, LS Mean (n = 36) (n = 90) (Patisiran −Placebo) P-Value Cardiac biomarkers NT-proBNP, Baseline score 155.9178.8 pmol/L Change from baseline 18 mos 227.2 12.5 −214.6 0.0024Troponin-1, Baseline score 0.11 0.12 mg/L Change from baseline 18 mos0.0 0.004 0.004 0.87 Echocardiogram LV wall Baseline score, mean 1.641.68 thickness, Change from baseline at 18 −0.007 −0.100 −0.093 0.0173cm mos Longitudinal Baseline score, mean −15.66 −15.13 Strain, % Changefrom baseline at 18 1.46 0.08 −1.37 0.0154 mos LV Mass, g Baselinescore, mean 264.5 275.48 Change from baseline at 0.63 −15.12 −15.75 0.15D546/18 mos LV ejection Baseline score, mean 62.2 60.0 fraction, %Change from baseline at 18 0.57 1.00 0.43 0.78 mos

Patients receiving patisiran showed a stabilization in NT-proBNP, ascompared to placebo patients (patisiran: 12.5 pmol/L increase, placebo:227.2 pmol/L increase). The difference in NT-proBNP between thepatisiran treated and the placebo patients was −214.6 pmol/L (p=0.0024).

NT-proBNP marker in cardiac subgoup Cardiac Subgroup Placebo Patisiran(N = 36) (N = 90) N % N % NT-proBNP >3000 pg/mL 24 25.0 80 10 at 18months* Progressors: NT-proBNP 12 58.3 38 21.1 increase ≥30% and >300pg/mL at month 18^(‡) Improvers: NT-proBNP 12 0 38 31.6 decrease ≥30%and >300 pg/mL^(‡) *Include only patients with non-missing NT-proBNP at18 months ^(‡)Include only patients with baseline NT-proBNP ≥650 ng/Land non-missing NT-pro BNP at 18 months

Improvements were also seen in LV (left ventricle) wall thickness andlongitudinal strain of patients receiving patisiran. LV wall thicknessdecreased 0.1 cm compared to baseline in patisiran treated patients,compared to only 0.007 cm in placebo patients (p=0.0173). LVlongitudinal strain also stabilized in patisiran patients, increasingonly 0.08% as compared to 1.46% in placebo patients (p=0.0154).

Example 5: Summary

In some embodiments, the methods described herein are used for treatinghereditary transthyretin-mediated amyloidosis (hATTR) withcardiomyopathy and polyneuropathy in a human patient in need thereof byadministering to the patient patisiran with a formulation as describedin Table 1 at a dose of 0.3 mg siRNA per kg body weight, wherein thepatisiran is administered intravenously once every 21 days or 3 weeks.The method results in a decrease in the modified Neuropathy ImpairmentScore (mNIS+7) composite neurological impairment score from thesubject's baseline score before the administration of patisiran.

In some embodiments, the methods described herein are used for treatinghereditary transthyretin-mediated amyloidosis (hATTR) withcardiomyopathy in a human patient in need thereof, the method comprisingadministering to the patient patisiran with a formulation as describedin Table 1 at a dose of 0.3 mg siRNA per kg body weight, wherein thepatisiran is administered intravenously once every 21 days or 3 weeks.

In some embodiments, the methods described herein are used for reducinga modified Neuropathy Impairment Score (mNIS+7) composite neurologicalimpairment score in a human patient having treating hereditarytransthyretin-mediated amyloidosis (hATTR) with cardiomyopathy andpolyneuropathy, the method comprising administering to the patientpatisiran with a formulation as described in Table 1 at a dose of 0.3 mgsiRNA per kg body weight, wherein the patisiran is administeredintravenously for once every 21 days or 3 weeks, wherein the methodresults in a decrease in the modified Neuropathy Impairment Score(mNIS+7) composite neurological impairment score from baseline asdetermined at 18 months, wherein baseline is the mNIS+7 score of thepatient before administration of patisiran.

In some embodiments, the method results in an improvement over baselinein one or more endpoints selected from the group consisting of a NorfolkQuality of Life Questionnaire-Diabetic Neuropathy (QOL-DN); a NIS-W; aRasch-built Overall Disability Scale (R-ODS); a 10-meter walk test; amodified body mass index (mBMI); a COMPASS-31 score. In someembodiments, the method results in an improvement in all of theendpoints. In some embodiments, the method results in an improvement ina Norfolk Quality of Life Questionnaire-Diabetic Neuropathy (QOL-DN);and a COMPASS-31 score and a 10-meter walk test.

In some embodiments, the patient is administered a premedication such asdexamethasone, oral paracetamol/acetaminophen, diphenhydramine,hydroxyzine, fexofenadine, cetirizine, ranitidine, famotidine, or otherIV histamine H1 or H2 receptor antagonists. In some embodiments, thepremedication is administered approximately one hour before thepatisiran. In some embodiments, the patient is further administered anoral daily dose of the USDA recommended daily allowance of vitamin A. Insome embodiments, the patient is also administered a tetramerstabilizer, such as tafamidis or diflunisal.

In some embodiments, the patient treated with the disclosed methods maybe Caucasian; may live in North America; may be 65 years old or older;may be male; may have FAP Stage I; may have FAP Stage II; may have abaseline mNIS+7 score between 8 and 165; may have a Val30 Met TTRmutation; may have one or more TTR mutations found in Table X; may haveechocardiographic evidence of cardiac amyloid involvement; and/or mayhave a history of prior long term TTR tetramer stabilizer use. In someembodiments, the administration of at least one drug is performed by thepatient. In other embodiments, the administration of at least one drugis performed by a medical professional.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

The invention claimed is:
 1. A method of treating cardiomyopathy in ahuman patient with transthyretin-mediated amyloidosis (TTR amyloidosis),the method comprising administering to the patient a patisiran drugproduct, wherein the patisiran drug product comprises an siRNA whichconsists of a sense strand comprising the nucleotide sequence5′-GuAAccAAGAGuAuuccAudTdT-3′ and an antisense strand comprising thenucleotide sequence 5′-AUGGAAuACUCUUGGUuACdTdT-3′, wherein A isadenosine, C is cytidine, G is guanosine, U is uridine, a is2′-O-methyladenosine, c is 2′-O-methylcytidine, g is2′-O-methylguanosine, u is 2′-O-methyluridine and dT is2′-deoxythymidine, wherein the siRNA is formulated in a compositioncomprising (6Z, 9Z, 28Z, 31Z)-heptatriaconta-6, 9, 28,31-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, andα-(3′-{[1,2-di(myristyloxy)propanoxy] carbonylamino}propyl)-ω-methoxy,polyoxyethylene (PEG2000-C-DMG), wherein the patisiran drug product isadministered intravenously once every 3 weeks at a dose of 0.3 mg siRNAper kg body weight, and the method results in stabilization orimprovement of a serum NT-proBNP concentration and/or a left ventricle(LV) strain and/or a LV wall thickness compared to baseline asdetermined before administration of the patisiran drug product.
 2. Themethod of claim 1, wherein the TTR amyloidosis is with cardiomyopathyand polyneuropathy, and wherein the patisiran drug product isadministered intravenously once every 3 weeks, wherein the methodresults in a decrease in the modified Neuropathy Impairment Score(mNIS+7) composite neurological impairment score from baseline asdetermined at 18 months, wherein baseline is the mNIS+7 score of thepatient before administration of the patisiran drug product.
 3. A methodfor stabilizing or improving a serum NT-proBNP concentration and/or aleft ventricle (LV) strain and/or a LV wall thickness in a human patienthaving transthyretin-mediated amyloidosis (TTR amyloidosis) withcardiomyopathy, the method comprising administering to the patient apatisiran drug product, wherein the patisiran drug product comprises ansiRNA which consists of a sense strand comprising the nucleotidesequence 5′-GuAAccAAGAGuAuuccAudTdT-3′ and an antisense strandcomprising the nucleotide sequence 5′-AUGGAAuACUCUUGGUuACdTdT-3′,wherein A is adenosine, C is cytidine, G is guanosine, U is uridine, ais 2′-O-methyladenosine, c is 2′-O-methylcytidine, g is2′-O-methylguanosine, u is 2′-O-methyluridine and dT is2′-deoxythymidine, wherein the siRNA is formulated in a compositioncomprising (6Z, 9Z, 28Z, 31Z)-heptatriaconta-6, 9, 28,31-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, andα-(3′-{[1,2-di(myristyloxy)propanoxy] carbonylamino}propyl)-ω-methoxy,polyoxyethylene (PEG2000-C-DMG), wherein the patisiran drug product isadministered intravenously once every 3 weeks at a dose of 0.3 mg siRNAper kg body weight, and wherein the method results in stabilization orimprovement of the serum NT-proBNP concentration and/or the leftventricle (LV) strain and/or the LV wall thickness, respectively,compared to baseline as determined before administration of thepatisiran drug product.
 4. The method of claim 2, wherein the changefrom baseline of the mNIS+7 score is −6.0 points.
 5. The method of claim2, wherein the decrease from baseline of mNIS+7 score is also determinedat 9 months.
 6. The method of claim 2, wherein the method results in animprovement over baseline in one or more neuropathy related clinicalendpoints selected from the group consisting of a. a Norfolk Quality ofLife Questionnaire-Diabetic Neuropathy (QOL-DN); and b. a NIS-W; and c.a Rasch-built Overall Disability Scale (R-ODS); and d. a 10-meter walktest (10-MWT); and e. a modified body mass index (mBMI); and f. aCOMPASS-31 score.
 7. The method of claim 6, wherein the method resultsin an improvement in all of the neuropathy related clinical endpoints.8. The method of claim 6, wherein the method results in an improvementin a Norfolk Quality of Life Questionnaire-Diabetic Neuropathy (QOL-DN)and a COMPASS-31 score and a 10-meter walk test.
 9. The method of claim6, wherein the method results in a serum percent TTR concentrationreduction in the patient compared to baseline as determined beforeadministration of the patisiran drug product.
 10. The method of claim 2,wherein the method results in stabilization or regression of a FAP stagein the patient compared to baseline as determined before administrationof the patisiran drug product.
 11. The method of claim 2, wherein themethod results in stabilization or regression of a PND score compared tobaseline as determined before administration of the patisiran drugproduct.
 12. The method of claim 2, wherein the method results in adecrease in an intra epidermal nerve fiber density in a skin biopsycompared to baseline as determined before administration of thepatisiran drug product.
 13. The method of claim 2, wherein the patientis administered the patisiran drug product for at least 12 months, 18months, 24 months, 30 months, or 36 months.
 14. The method of claim 3,wherein the patient is in need of treatment for transthyretin-mediatedamyloidosis (TTR amyloidosis) with cardiomyopathy and the method resultsin an improvement or a stabilization of a cardiac marker and/or anechocardiogram parameter compared to baseline as determined beforeadministration of the patisiran drug product.
 15. The method of claim14, wherein the cardiac marker is a serum NT-proBNP concentration andthe echocardiogram parameter is a left ventricle (LV) strain or a LVwall thickness.
 16. The method of claim 1, further comprisingadministering to the patient the following premedications:dexamethasone, oral paracetamol/acetaminophen, diphenhydramine, andranitidine.
 17. The method of claim 1, further comprising administeringto the patient the following premedications: a. IV dexamethasone 10 mg,or equivalent; and b. oral paracetamol/acetaminophen 500 mg, orequivalent; and c. IV histamine H1 receptor antagonist (H1 blocker):diphenhydramine 50 mg, or equivalent other IV H1 blocker or hydroxyzine25 mg or fexofenadine 30 or 60 mg PO or cetirizine 10 mg PO; and d. IVhistamine H2 receptor antagonist (H2 blocker): ranitidine 50 mg orfamotidine 20 mg, or equivalent other H2 blocker dose.
 18. The method ofclaim 16, wherein the premedications are administered approximately onehour prior to each patisiran drug product administration.
 19. The methodof claim 1, further comprising administering to the patient an oraldaily dose of the USDA recommended daily allowance of vitamin A.
 20. Themethod of claim 1, further comprising administering a tetramerstabilizer, wherein the tetramer stabilizer is tafamidis or diflunisal.21. The method of claim 1, wherein the patient a. is Caucasian; and/orb. lives in North America; and/or c. is 65 years old or older; and/or d.is male; and/or e. has FAP Stage I; and/or f. has FAP Stage II; and/org. has a baseline mNIS+7 score between 8 and 165; and/or h. has a Val30Met TTR mutation; and/or i. has one or more TTR mutations found in TableX; and/or j. has echocardiographic evidence of cardiac amyloidinvolvement; and/or k. has a history of prior long term TTR tetramerstabilizer use.
 22. The method of claim 1, wherein administration isperformed over 80 minutes.
 23. The method of claim 1, wherein baselineis an average.
 24. The method of claim 1, wherein the siRNA isformulated in a composition comprising 13.0 mg/mL of (6Z, 9Z, 28Z,31Z)-heptatriaconta-6, 9, 28, 31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 3.3 mg/mL of1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 6.2 mg/mL ofcholesterol, and 1.6 mg/mL of α-(3′-{[1,2-di(myristyloxy)propanoxy]carbonylamino}propyl)-ω-methoxy, polyoxyethylene (PEG2000-C-DMG). 25.The method of claim 3, wherein the siRNA is formulated in a compositioncomprising 13.0 mg/mL of (6Z, 9Z, 28Z, 31Z)-heptatriaconta-6, 9, 28,31-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA), 3.3 mg/mLof 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 6.2 mg/mL ofcholesterol, and 1.6 mg/mL of α-(3′-{[1,2-di(myristyloxy)propanoxy]carbonylamino}propyl)-ω-methoxy, polyoxyethylene (PEG2000-C-DMG). 26.The method of claim 1, wherein the TTR amyloidosis is a hereditary TTRamyloidosis.
 27. The method of claim 3, wherein the TTR amyloidosis is ahereditary TTR amyloidosis.